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Abstract

Arterial baroreflex function diminishes with age, but whether cardiopulmonary vagal reflexes are similarly altered with physiological aging has not been fully elucidated. In this study, predominantly cardiac high pressure mechanoreceptor-activated (ramp baroreflex) and cardiopulmonary chemoreceptor-activated (von Bezold-Jarisch reflex) vagal reflexes in conscious, instrumented rats were impaired by 30% to 40% (P<0.05) in 24-month-old (n=12) compared with 6-month-old rats (n=12). To determine whether this is a restorable deficit, the influence of atrial natriuretic peptide (ANP), either by infusion or blockade of its breakdown, was studied. ANP infusion was previously shown to enhance Bezold-Jarisch reflex and ramp baroreflex bradycardia in young adult rats. The present study confirmed that vagal reflex augmentation by ANP (50 pmol/kg per minute) also occurs in old rats (increased by 60±18% (Bezold-Jarisch reflex) and 91±15% (ramp baroreflex; P<0.05). Direct vagal stimulation in anesthetized animals showed that the target for ANP was not the cardiac vagus itself in old rats (n=7), although in young rats only, we confirmed the published finding that ANP enhances vagal bradycardia (by 58±14%, n=7). Neutral endopeptidase 24.11 degrades ANP and several other peptides. The neutral endopeptidase inhibitor candoxatrilat (5 mg/kg per day IV for 7 to 9 days) restored vagal reflex bradycardia in old rats (n=6) to levels similar to those in young neutral endopeptidase inhibitor-treated rats (n=6). Impaired cardiopulmonary vagal reflex control of heart rate is thus a feature of normal aging, and this deficit may be ameliorated by either ANP infusion or chronic neutral endopeptidase inhibition.

Diminished arterial baroreflex control of heart rate (HR) is a well-documented feature of aging (see reviews1,2), but surprisingly few studies have examined the effect of aging on reflexes originating in the heart. It is generally accepted that low-pressure cardiopulmonary reflexes are less effective with advancing age and that this may play a role in the decreased ability of the elderly to regulate blood volume and maintain blood pressure during gravity challenges.3,4 Whether other cardiocardiac vagal reflexes such as the cardiopulmonary chemoreflex (von Bezold-Jarisch [BJR]) or those activated by high-pressure ventricular stretch receptors (ramp baroreflex) are also compromised with age is not clear. The BJR is thought to be cardioprotective (promoting reflex bradycardia, hypotension, and vasodilatation) and is activated by 5-HT3 receptors on ventricular vagal afferents.5 Davidow and colleagues concluded that the BJR in conscious 14- and 24-month-old rats was reduced compared with 6-month-old rats,6 whereas Sakima and colleagues7 reported that the BJR was not diminished in older (16- to 20-month-old) rats.

The potential for cardioprotection by enhancing parasympathetic activity has long been recognized and this avenue is underexploited by current therapies.8 In young animals, acute administration of atrial natriuretic peptide (ANP) enhances not only the BJR,9,10 but also the reflex bradycardia after cardiopulmonary high-pressure baroreceptor activation by the rapid “ramp” method,9,11,12 an effect still evident in sinoaortically denervated rats.12 The influence of ANP on cardiopulmonary vagal afferent reflexes in aging has not been previously reported. As a peptide, ANP has limited clinical potential due to its rapid gastrointestinal inactivation when administered orally, whereas inhibiting the enzyme responsible for the metabolism of natriuretic peptides (neutral endopeptidase [NEP], EC 24.11) potentiates renal and hemodynamic effects of the peptide clinically and experimentally.13,14

The present study therefore determined if acute increases in synthetic ANP, and chronic increases in endogenous natriuretic peptides after NEP inhibition, enhance cardiopulmonary vagal reflex function in young and old rats. The study aims were: (1) to assess whether cardiopulmonary vagal reflexes are diminished with age in conscious rats; (2) to test whether the effect of acute infusions of ANP on these cardiopulmonary vagal reflexes is preserved in aged rats; (3) to determine whether vagal efferent neurotransmission is a target for this action of ANP; and (4) to examine whether chronic administration of a NEP inhibitor (candoxatrilat) influences vagal reflexes in old or young rats.

Methods

General

This study was approved by the Howard Florey Institute Animal Ethics Committee and performed in accordance with the Australian Guidelines for Care and Use of Laboratory Animals. Cardiac vagal reflexes were examined in male Munich-Wistar (Animals Resources, Perth, Australia) conscious rats, chronically instrumented with arterial and venous catheters, and allocated to 1 of 4 groups: 6-month-old rats (n=12, age range 6 to 7 months), 24-month-old rats (n=12, age range 23 to 26 months), NEP inhibitor-treated 6-month-old rats (n=6, age range 6 to 7 months), and NEP inhibitor-treated 24-month-old rats (n=6, age range 23 to 25 months). Nonrecovery, vagal stimulation experiments were carried out in further anesthetized 6-month-old (n=7) and 24-month-old (n=7) Munich-Wistar rats.

Surgical Preparation for Chronic Instrumentation

Surgical procedures were the same as those reported previously,9 except that the general anesthesia used for instrumentation was isoflurane (2%). Briefly, 2 vascular cannulae were implanted in each animal with the free ends of the cannulae tunnelled subcutaneously and exteriorized behind the neck. The cannula in the abdominal aorta was used for measuring arterial blood pressure and HR. The other cannula in the right jugular vein (and positioned near the right atrium) contained a triple lumen, which allowed simultaneous infusions of vasoactive drugs or ANP separately into each lumen. This cannula was also used for NEP inhibitor treatment.

NEP Inhibitor Treatment in Conscious Rats

Candoxatrilat (UK 73,967; Pfizer, Sandwich Laboratories, Kent, UK), a specific inhibitor of NEP, was administered daily (5 mg/kg per day intravenously in 0.5 mL saline over a 1-hour period) for 7 to 8 days beginning 1 day after instrumentation surgery and ending on the day of reflex testing, immediately before the start of the experiment. Candoxatrilat is the active product of the orally active Candoxatril, which has been used in numerous studies in humans15,16; a lower dose of Candoxatrilat (3 mg/kg) was reported to inhibit renal NEP binding in rats for up to 24 hours.17

Experimental Protocol for Reflex Measurements

Experiments were performed in conscious, unrestrained rats 1 week after instrumentation with an aged rat always paired with a younger one on a given day. At the start of each experiment, the arterial cannula was connected to a transducer (Cobe, Lakewood, Colo) and HR was measured using a tachograph (Baker Institute, Melbourne, Australia) triggered from the phasic blood pressure signal. Arterial pressure and HR were continually recorded at a sampling rate of 200 Hz using the AcqKnowledge data acquisition system (Biopac Systems, Golenta, Calif), which calculated mean arterial pressure (MAP). Cardiopulmonary vagal reflexes were tested in the presence of ANP (50 pmol/kg per minute intravenous infusion; Bachem AG, Bubendorf, Switzerland) or saline vehicle (390 μL/h) in alternate order. See the online data supplement at http://hyper.ahajournals.org for additional details.

HR Reflex Techniques

HR reflex testing methods in rats have been described previously in detail.11,12,18,19 Briefly, (1) ramp baroreflex responses were assessed after rapid MAP increases of approximately 50 mm Hg evoked by methoxamine (100 μg/kg intravenously; Sigma Chemical) aiming for a similar rate of MAP rise across all animals (approximately 20 mm Hg/s). Linear regression analysis was applied to the progressive HR responses to MAP changes. For full details, see Figure S1). Three replicate ramp tests were performed in each rat in the absence and presence of ANP, allowing full baseline recovery between tests; (2) BJR bradycardia and hypotension were measured to 3 bolus doses of serotonin (5-HT; 2 to 18 μg/kg intravenously; Sigma Chemical). The 3 5-HT doses were individually chosen for each rat to give threshold, intermediate, and submaximal responses; once chosen, they were maintained in that animal for tests both in the absence and presence of ANP. At least 10 minutes were allowed between successive 5-HT doses. For further details, see Figure S2.

ANP Measurements

Blood samples were collected from conscious rats after HR reflex testing had been completed and while ANP or saline vehicle was still being administered. The samples were centrifuged and the plasma was stored at −20°C until the time of analysis by radioimmunoassay (for details, see the data supplement). At the end of experiments in 6 young and 5 old rats, hearts were collected immediately after barbiturate overdose (Euthatal; pentobarbitone sodium, 350 mg/mL IV; Rhone Merieus). Atria and ventricles were separated, weighed, fresh–frozen in liquid nitrogen, and stored at −20°C for subsequent measurement of ANP content using the radioimmunoassay described for plasma.

Vagal Stimulation Experiments

Under general anesthesia (2% isoflurane), both cervical vagi were cut and the right vagus was prepared for stimulation. Propranolol (1 mg/kg, repeated as required; Sigma Chemical) was given to block sympathetic actions on the heart. The caudal end of the right vagus was stimulated with 10-second supramaximal trains at a range of frequencies, and the maximum increase in cardiac interval was measured in each case. Stimulus frequency–response relationships were measured before and at least 20 minutes after the onset of ANP infusion (50 pmol/kg per minute IV). At the completion of the experiment, the rat was euthanized with an overdose of barbiturate. For full details, see the online data supplement.

Data Analysis

The effects of age on cardiopulmonary reflex bradycardia and hypotension were determined from 2-way analysis of variance with repeated measures across doses of 5-HT (BJR) or replicate baroreflex ramps in untreated (n=12 in each group) or in NEP inhibitor-treated rats (n=6 in each group). The effect of ANP on vagal reflexes was determined by comparing the within-animal bradycardic responses to the same doses of 5-HT (BJR) or triplicate ramps (baroreflex) without and during ANP infusion by 2-way analysis of variance with repeated measures with a Bonferroni adjustment of the α for multiple comparisons. To determine whether the ANP effect was different between age groups, the additional effect of ANP (ie, the difference between reflex bradycardia during baseline [vehicle infusion] and during ANP infusion) in young and old rats was compared by 2-way analysis of variance.

The effects of age and ANP infusion on the efferent cardiac vagal activity were determined from the linear regression analysis of the relationship between stimulation frequency (up to 20 Hz) and the bradycardic response at each frequency step. Slopes were compared with 2-way analysis of variance. Baseline hemodynamics or ANP levels were analyzed for the effects of age or NEP inhibitor treatment by one-way analysis of variance. For all tests, P<0.05 was taken as the level of significance. Data are mean±SEM unless otherwise stated.

Results

Body Weight and Resting Hemodynamics in Young and Old Rats

Twenty-four-month-old Munich-Wistar rats were approximately 12% heavier than 6-month-old rats (461±12 g versus 411±5 g, respectively, P<0.05). NEP inhibitor treatment did not affect body weight in either old (467±19 g) or young (419±10 g) rats. There was no significant difference in resting MAP or HR between old and young rats (113±2 versus 114±2 mm Hg and 310±3 versus 309±4 beats/min, respectively; n=12 in each group).

Cardiopulmonary Vagal Reflexes Were Attenuated in Old Rats

von Bezold-Jarisch Reflex

In 6-month-old rats, the 3 doses of 5-HT that produced threshold, intermediate, and submaximal reflex bradycardia were 4.6±0.3, 7.9±0.7, and 11.2±0.9 μg/kg with an average dose of 8.0±0.6 μg/kg. Similar doses of 5-HT were administered to 24-month-old rats with an average dose of 5-HT of 7.7±0.7 μg/kg. Bradycardic responses across the 3 doses of 5-HT were significantly (P<0.05) attenuated in old compared with young rats (Figure 1A). The mean reflex change in heart period across all doses was 228±24 ms in young rats and 155±23 ms in old rats, a 32% decline with age. There was no significant difference in hypotension to 5-HT doses between age groups (−11±5 mm Hg in old versus −14±5 mm Hg in young rats).

Figure 1. Effect of age and NEP inhibition on BJR (bradycardic response to threshold, intermediate, and submaximal doses of 5-HT) in conscious 6-month-old (open symbols) and 24-month-old (gray symbols) rats. A, Untreated young (n=12) and old (n=12) rats; (B) NEP inhibitor-treated young (n=6) and old (n=6) rats. Symbols are mean±SED, where SED is between-animal SE of the difference for comparison between age groups. *P<0.05 (comparison across all doses).

Ramp Baroreflex

Methoxamine-induced “ramp” blood pressure rises in each age group were similar (19±1 mm Hg/s in old versus 22±1 mm Hg/s in young rats), and regression analyses relating change in HR to change in MAP all showed r2 values >0.93. Mean ramp gain in 6-month-old rats was −2.12±0.21 beats/min per mm Hg (Figure 2A), similar to previous measurements in 3-month-old animals,12,18 whereas in 24-month-old rats, it was −1.30±0.22 beats/min per mm Hg (Figure 2B, P<0.05), representing a 39% decline with age.

Is the Effect of ANP Infusion Preserved in Old Rats?

Baseline Hemodynamics

Acute ANP infusion reduced resting MAP in both old (−9±4 mm Hg) and young (−7±2 mm Hg) rats (n=12, P<0.05 for both groups). Baseline HR was unaffected by ANP infusion in either group.

von Bezold-Jarisch Reflex

In young rats, ANP significantly enhanced the reflex increase in heart period from 228±24 to 330±37 ms (within-animal comparison over all doses, P<0.05). In old rats, ANP also enhanced the reflex increase in heart period from 155±24 to 248±31 ms (P<0.05). The reflex changes to 5-HT doses before and after ANP are compared in Figure 3. The accompanying reflex hypotension was not different in the presence of ANP (−12±5 mm Hg in old and −18±8 mm Hg in young rats) compared with saline infusion (see previously) in either age group. For further details, see Figure S3A.

Figure 3. The additional effect of ANP infusion (50 pmol/kg per min) on the BJR in young (open circles, n=12) and old (gray circles, n=12) rats. The “baseline” reflex measurements are those shown in Figure 1A. Symbols are bradycardic responses to threshold, intermediate, and submaximal doses of 5-HT during saline infusion (CONTROL, x-axis) vs the bradycardic responses to the same doses of 5-HT during ANP infusion (WITH ANP, y-axis). Dotted line represents the relationship if no effect of ANP. HP indicates heart period. Symbols are mean±SEM, estimate of between-animal variation. The ANP effect is not different between ages.

Ramp Baroreflex

In both age groups, the methoxamine-induced rates of rise in MAP during ANP infusion were similar to control conditions. In 6-month-old rats, ANP infusion significantly (P<0.05) increased ramp baroreflex gain to −3.20±0.24 beats/min per mm Hg (Figure 2C; a 51% increase) in line with findings on 3-month-old rats.12,18 In old rats, ANP also significantly (P<0.05) enhanced the ramp baroreflex to −2.47±0.31 beats/min per mm Hg (Figure 2D, a 91% increase). For further details, see Figure S3B.

Cardiac Vagal Neurotransmission

Unlike in the conscious state (see previously), resting HR (277±10 beats/min) of old rats under anesthesia was significantly (P<0.05) lower than in the younger rats (320±9 beats/min). This difference was maintained after vagotomy and propranolol, showing that old rats had a lower intrinsic heart rate than young rats (241±7 versus 282±14 beats/min; P<0.05) as is the case for older humans.20 The bradycardia response to efferent vagal stimulation at up to 20 Hz closely fit a linear relationship with r2 values >0.96 in all animals. This relation showed no significant difference in slope between old and young rats (−3.97±0.64 versus −3.13±0.40 beats/min per Hz). ANP infusion significantly (P<0.05) enhanced the slope of the relationship in young rats (to −4.74±0.49 beats/min per Hz; Figure 4, left graph) but not in old rats (Figure 4, right graph).

Baseline Hemodynamics

Chronic treatment with a NEP inhibitor did not alter the baseline hemodynamics of young (111±2 mm Hg and 318±9 beats/min, n=6) or old rats (116±1 mm Hg and 312±8 beats/min, n=6) compared with levels in untreated rats, shown previously.

von Bezold-Jarisch Reflex

Chronic NEP inhibition removed the BJR deficit in old rats compared with young rats (Figure 1B). The mean doses of 5-HT used to evoke the BJR were similar in old and young NEP inhibitor-treated rats (8.2±0.7 μg/kg and 7.5±0.8 μg/kg, respectively) and were similar to doses of 5-HT given to untreated animals of both ages (see previously). The hypotension in response to 5-HT was similar in old and young NEP inhibitor-treated rats (−11±7 mm Hg and −9±3 mm Hg, respectively).

Ramp Baroreflex

Similar to its effects on the BJR, chronic NEP inhibition reversed the effect of age on the high pressure cardiac baroreflex such that there was no difference in ramp gain in old compared with young NEP inhibitor-treated rats (−1.95±0.28 versus −1.86±0.14 beats/min per mm Hg; Figure 2E–F). The rates of rise in MAP in these tests were not perfectly matched, however, being greater in young than in old NEP-inhibitor treated rats (28±3 mm Hg/s versus 19±1 mm Hg/s; P<0.05).

Effect of Age on Plasma and Heart ANP Levels

Plasma

Resting plasma ANP levels tended to be higher (P=0.06) in 24-month-old rats (80±11 fmol/mL, n=7) compared with 6-month-old rats (46±10 fmol/mL, n=10). Acute ANP infusion increased these levels by 85±34 fmol/mL in old rats and by 130±52 fmol/mL in young rats. Chronic NEP inhibitor treatment did not significantly change resting plasma ANP levels in either age group (65±14 fmol/mL in old rats, n=5; 67±5 fmol/mL in young rats, n=6). Infusions of ANP into NEP inhibitor-treated animals raised plasma ANP levels by 273±68 fmol/mL in old rats and by 327±144 fmol/mL in young rats. This greater increase in plasma ANP (approximately 3-fold compared with untreated rats) was significant (P<0.05, n=11).

Heart

ANP levels in heart tissues were not different between 24-month-old and 6-month-old rats. Left atrial tissue ANP content was 198±68 nmol/g wet weight in old rats (n=5) and 231±21 nmol/g wet weight in young rats (n=6). Right atrial tissue ANP content was 391±96 nmol/g wet weight in old rats and 411±64 nmol/g wet weight in young rats. Combined ventricular tissue ANP concentration was 803±296 pmol/g wet weight in old and 775±217 pmol/g wet weight in young rats.

Discussion

The present study demonstrates that old rats (24-month-old) have a deficit in the ramp baroreflex bradycardia (approximately 39%) and a quantitatively similar deficit in the BJR (approximately 32%) compared with younger but mature adults (6-month-old). For the first time, this study provides clear evidence that manipulation of the natriuretic peptide system, either acutely with infusion of ANP or chronically (although less specifically) with NEP inhibition, improves cardiopulmonary vagal reflex function in old rats.

Munich-Wistar rats were chosen for these studies because they do not become obese with age with ad libitum feeding, unlike many other rat strains.21,22 Although not possessing longitudinal data on cardiopulmonary reflex changes, our present and previous findings in Munich-Wistar rats suggest that age-associated deficits in HR reflexes progress throughout the lifetime of the animal. Cardiac baroreflex sensitivity falls between 3 and 6 months of age12 (present study) and diminishes further still between 6 and 24 months of age. For the present study, 6-month-old rats were chosen as the younger control because at this age, they have reached full maturity, thereby minimizing the potentially confounding influences of body size and growth that occur during adolescence. The use of chronic instrumentation allowed all reflex studies to be done in awake, quiet, and unstressed animals, thereby ensuring that the cardiovascular reflex measurements were carried out under optimal conditions.

What is the cause of reduced cardiopulmonary reflex function with age? Neural deficits may contribute to diminished autonomic reflex function with age. Efferent autonomic dysfunction is generally not favored as an explanation for the loss of vagal tone and responsiveness.23 End-organ autonomic receptors appear to be intact,3 and our present results confirmed that age caused no diminution in cardiac responsiveness to vagal stimulation24 (Figure 4). Age-related loss of cardiac reflex function may therefore result from changes in (1) the sensory receptors; (2) the afferent nerves; or (3) central nervous processing (as occurs for arterial baroreflexes25). Our present data cannot distinguish which nor can they tell us whether natriuretic peptides are involved (see subsequently).

Regardless of the underlying mechanisms of the age-related deficit, the ameliorating effect of systemic ANP would be due to an action outside the blood–brain barrier at the level of the vagal afferents9–12 or possibly the sensory circumventricular organs.26 In young rats only, we confirmed Atchison and Ackermann’s observation27 that ANP may enhance cardiac responsiveness to vagal efferent stimulation. This effect was not present in old rats, yet they showed reflex enhancement by ANP comparable with that of young rats. In old rats at least, the major site of ANP’s action cannot be efferent. Interestingly, we recently found that under similar conditions, BNP infusion in young adult Wistar rats enhanced the BJR, yet had no effect on vagal efferent responsiveness.28

Because additional ANP restored the vagal reflex deficit in old rats, we considered the hypothesis that normal circulating levels of ANP may decline with age. However, as other studies have shown in different rat strains,21,29 and indeed in humans,30,31 plasma ANP levels in old Munich-Wistar rats were increased approximately 2-fold compared with younger rats. These elevated plasma levels are most likely due to decreased clearance31 because cardiac levels of ANP were not enhanced in old rats.

So why do the elevated circulating ANP levels not sustain normal reflex bradycardic activity in old rats? To our knowledge, alterations in natriuretic peptide receptor expression have not been examined in aging. One possibility is that some reduction in natriuretic peptide receptor number or action prevents the endogenous ANP (and other natriuretic peptides) from maintaining cardiac reflex function in old rats. Against this argument is the observation that exogenous ANP, at doses that raise plasma levels approximately 10-fold, are as effective in old as in young rats. This does not, however, rule out the possibility that old rats have some modest deficit in the basal level of natriuretic peptide signaling, which we have shown to be necessary for the full expression of the BJR.19 Many other mechanisms could underlie the reduced reflex responsiveness of older rats, however, and the issue awaits direct study.

The finding that endogenous ANP levels did not rise after 1 week of Candoxatrilat treatment is not uncommon. Other studies have shown that with chronic NEP inhibitor treatment, ANP levels re-establish at a steady-state level where release falls, compensating for the reduced clearance.32,33 NEP inhibitor treatment markedly reduced the breakdown of exogenous ANP as demonstrated by potentiated plasma ANP levels measured in both age groups of rats when the peptide was infused.

Although candoxatrilat is a specific inhibitor of NEP, the enzyme acts on a number of substrates in addition to ANP and other natriuretic peptides. Our focus was on ANP in the present study, but BNP, and to a lesser extent CNP, have similar actions on cardiopulmonary vagal reflex function10,11 so the effects of NEP inhibition may involve all natriuretic peptides. Chronic NEP inhibition may also act through other NEP substrates, which include adrenomedullin, bradykinin, substance P, and endothelin.34–36 There is little evidence that adrenomedullin or endothelin influences cardiopulmonary reflex function, but bradykinin and substance P have been shown to enhance the BJR.37,38 Thus, NEP inhibition could enhance cardiopulmonary vagal reflexes in old rats also by non-natriuretic peptide actions.

In summary, cardiac vagal afferent-driven baro- and chemoreflex functions were reduced in elderly Munich-Wistar rats. These reflexes remained sensitive to the enhancing action of ANP, however, and chronic NEP inhibition was able to abolish the deficit in old rats compared with young adults. Enhanced availability of endogenous natriuretic, and perhaps other peptides, presumably underlies the restorative effect of NEP inhibition.

Perspectives

The vagal reflexes studied here may be considered to be cardioprotective and would act to unload the heart during periods of ischemia or excessive arterial pressure. Natriuretic peptides enhance those actions, an effect that survives into old age when it may be most needed. Furthermore, natriuretic peptide enhancement of reflexly activated parasympathetic activity may shift the sympathovagal balance to prevent or delay adverse cardiovascular events and mortality caused by increased sympathetic drive in apparently healthy elderly humans.

Acknowledgments

We thank Tony Dornom for his expert technical assistance with surgical preparation of the animals and Greg Thomas and Tom Vale for the long-term care and maintenance of the animals. Candoxatrilat was a generous gift from Pfizer Global Pharmaceuticals, Australia.

Source of Funding

This study was supported by a block grant from the National Health and Medical Research Council of Australia (No. 983001).